Disproof of the Structures and Biosynthesis of Ergoynes, Gs-Polyyne–l-Ergothioneine Cycloadducts from Gynuella sunshinyii YC6258

Some bacteria produce "bacterial polyynes" bearing a conjugated C≡C bond that starts with a terminal alkyne. Ergoynes A and B have been reported as sulfur-containing metabolites from Gynuella sunshinyii YC6258. These compounds were thought to be formed by cycloaddition between a bacterial polyyne (named Gs-polyyne) and l-ergothioneine. The biosynthetic gene clusters (BGCs), which may contribute to their synthesis, were present in the YC6258 genome. The biosynthetic origin of Gs-polyyne is interesting considering its rare 2-isopentyl fatty acyl skeleton. Here, the structures and biosynthesis of Gs-polyyne and ergoynes were verified by analytical, chemical, and genetic techniques. In the YC6258 extract, which was prepared considering their instability, Gs-polyyne was detected as a major LC peak, and ergoynes were not detected. The NMR data of the isolated Gs-polyyne contradicted the proposed structure and identified it as the previously reported protegenin A. The expression of Gs-polyyne BGC in Escherichia coli BL21(DE3) also yielded protegenin A. The cyclization between protegenin A and l-ergothioneine did not proceed during sample preparation; a base, such as potassium carbonate, was required. Overall, Gs-polyyne was identified as protegenin A, while ergoynes were determined to be artifacts. This cyclization may provide a derivatization to stabilize polyynes or create new chemical space.